biotinylated isotype-matched control immunoglobulin g (igg  (Thermo Fisher)

Journal: Journal of Extracellular Biology

Article Title: Hepatocyte‐derived extracellular vesicles regulate liver regeneration through a negative feedback mechanism

doi: 10.1002/jex2.70023

Figure Lengend Snippet: HD‐EVs blocks proliferation of mouse hepatocytes (AML‐12) in vitro. (a) The schematic illustration of the in vitro experiments. (b) Flow cytometry analysis for EVs double‐labeled with DiB/AF647‐conjugated Anti‐mASGR1 in the GP‐EVs, after immunoprecipitation with biotinylated Anti‐ASGR1 Ab (NH‐EVs), and after immunoprecipitation with biotinylated rabbit IgG isotype (r‐IgGi) control. (c) EVs uptake assay for the GP‐EVs and for the NH‐EVs. Twenty‐four hours after seeding, AML‐12 cells were incubated for different periods of time with two different types of DiL‐labelled EVs at a final concentration of 2 × 10 11 particles/mL. At the end of the incubation time, the cells were washed, fixed, and stained with mounting media with DAPI. Scale bar = 200 µm. (d) Cell cycle analysis for AML‐12 cells incubated with vehicle (PBS) and with different concentrations of GP‐EVs extracted from mouse plasma. As negative controls: (i) AML‐12 cells and GP‐EVs were preincubated for 2 h with 5 µg/mL of Heparin; (ii) AML‐12 cells were incubated with NH‐EVs. (e) Graph representation of the cell cycle analysis shown in (d). (f) Western blot analysis for expression of Cyclin D1, PCNA, and GAPDH in lysates of AML‐12 cells treated as shown in (d). Shown is representative data from three independent experiments. (g) Graphs with quantification of the Western blot analysis as shown in (f). n = 3 independent experiments. EVs, extracellular vesicles; GP‐EVs, global plasma EVs; NH‐EVs, non‐hepatocyte EVs; PBS, phosphate‐buffered saline.

Article Snippet: As a negative control, EVs precipitation was performed with biotinylated rabbit IgG isotype control (Cell Signalling Technology, Danvers, MA, USA).

Techniques: In Vitro, Flow Cytometry, Labeling, Immunoprecipitation, Control, Incubation, Concentration Assay, Staining, Cell Cycle Assay, Clinical Proteomics, Western Blot, Expressing, Saline